In this revised renewal application we are proposing to exploit heteroduplex tracking assays we have developed to assess HIV-1 population complexity in the study of acute HTV-1 infection. We will work with human samples collected from two separate cohorts: the STAT cohort in North Carolina (from statewide VCT sites) and a STD clinic cohort in Lilongwe, Malawi. For both of these cohorts all samples are screened in real time for acute HIV-1 infection (seronegative/RNA+). Subjects in the Malawi cohort are followed longitudinally for 16 weeks. In initial studies of two other cohorts, largely of MSM, we have found that 50% of subjects are infected with multiple variants. We have also studied primary infection of macaques with SIV where we documented a stable V1/V2 env gene population over an 8-12 week period, establishing the timing over which human samples can be collected and retain information about the complexity of the infecting virus. Working with samples from these two cohorts we will: i) document the complexity of the transmitted virus population during homosexual and heterosexual transmission involving subtype B HIV-1; document the complexity of the transmitted virus population during heterosexual transmission involving subtype C HIV-1 for men and women; ii) use disparate HTA patterns of more conserved regions as a screening method for identifying dual infections during the acute infection period; iii) use the longitudinal samples from the Malawi cohort to examine the early events in the evolution of the surface Env protein; and iv) use these same longitudinal samples to explore the feasibility and utility of carrying out bulk sequence analysis of amplicons representing the viral proteome to find rapidly fixed changes suggestive of strong CTL selection during the resolution of viremia. These studies will provide insights into the molecular genetics of viral transmission in settings in the rural US and in southern Africa, and use the earliest events of virus evolution to infer the nature of the initial virus-host interaction and the sites of selection of the adaptive immune response. Identifying sites in the viral genome that are under strong early selective pressure may reveal those sites that are the most relevant targets for vaccine design. Understanding the complexity of the transmitted virus is central to understanding the mechanism of penetration of the mucosal layer and will inform strategies for microbicide development.